ES2209492T3 - COLORS OF ENERGY TRANSFER. - Google Patents

Abstract

Energy transfer dyes, their preparation, and their use as labels in biological systems is disclosed. The dyes are preferably in the form of cassettes which enable their attachment to a variety of biological materials. The dyes and the reagents that can be made from them offer a wide variety of fluorescent labels with large Stokes' shifts enabling their use in a variety of fluorescence applications over a wide range of the visible spectrum. The energy transfer dyes may be of the formula:<BR> <BR> D<SB>1</SB>- L<SB>1</SB>-A-L<SB>2</SB>- D<SB>2</SB><BR> <BR> wherein<BR> <SL> <LI>D<SB>1</SB> is a first dye moiety suitable as an acceptor or donor in an energy transfer arrangement; <LI>D<SB>2</SB> is a second dye moiety suitable as a donor or acceptor in an energy transfer arrangement with the first dye; </SL> ```A comprises 5-20 linked atoms, wherein the linked atoms contain at least one aryl or heteroaryl group and wherein the atoms are independently selected from the group consisting of carbon, nitrogen, sulfur, oxygen, phosphorus;<BR> ```L<SB>1</SB> and L<SB>2</SB> comprise a group of linked atoms and L<SB>2</SB> is adapted for attaching to a biological target. The following are specifically claimed 5-FAM-Phe-5-REG-11-ddUTP, 5-FAM-Phe-5-REG-11-ddCTP, 5-FAM-Phe-5-REG-11-ddATP, 5-FAM-Phe-5-REG-11-ddGTP, 5-FAM-Phe-5-ROX-11-ddCTP, 5-FAM-Phe-5-ROX-11-ddUTP, 5-FAM-Phe-5-ROX-11-ddATP, 5-FAM-Phe-5-ROX-11-ddGTP, 5-FAM-Phe-5-TAMARA-11-ddGTP, 5-FAM-Phe-5-TAMARA-11-ddATP, 5-FAM-Phe-5-TAMARA-11-ddCTP, 5-FAM-Phe-5-TAMARA-11-ddUTP, 5-FAM-Phe-5-R110-11-ddATP, 5-FAM-Phe-5-R110-11-ddUTP, 5-FAM-Phe-5-R110-11-ddGTP, and 5-FAM-Phe-5-R110-11-ddCTP. An energy transfer cassette may be of the formula: <EMI ID=1.1 HE=19 WI=43 LX=1009 LY=1917 TI=CF> <PC>```wherein F<SP>1</SP> is a first dye suitable as an acceptor or donor in an energy transfer arrangement;<BR> ```F<SP>2</SP> is a second dye that is suitable as a donor or acceptor in an energy transfer arrangement with the first dye;<BR> ```B is one or more alkyl groups containing from 1 to 30 atoms, wherein said atoms comprise one or more -NH-, -O-, -CH=CH-, -C I C- and phenylenyl groups, and wherein said atoms are selected from the group consisting of C<SB>1-4</SB> linear or branched alkyl, hydroxyl, halo, or phenyl;<BR> ```M<SP>3</SP> comprises hydrogen or a reactive or functional group adapted for attaching the energy transfer dye to a target material; and<BR> ```D comprises an atom or group adapted for attaching F<SP>2</SP> to the linker group B.

Description

Energy transfer dyes

The present invention relates to a new class of energy transfer dyes, their preparation and their use as markers of biological systems.

Background of the invention

Certain techniques are described below. relevant, none of which is admitted as prior art in the attached claims.

Various methodologies are available for visualization of cells or molecules in cells and for measurement of analyte concentrations in fluids. The microscopy of fluorescence uses fluorescent dyes, generally associated with specific probes, such as antibodies, for the location of proteins and complexes in cells.

For the measurement of analyte concentrations, the detection of an analyte of interest, the determination of particular sequence of a nucleic acid molecule, immunoassays and various hybridization methods have become Popular methods in the last 40 years. The radioimmunoassays are developed because the high specific activity of the radionuclides allowed the measurement of very low concentrations of analyte. However, due to related concerns with the environment and human health, the use of radionuclides In immunoassays it is being less popular. The use of enzymes in immunoassays to amplify a signal has been a very breakthrough important in the field of immunoassays because its use does not involve environmental or human health hazards or risks. Without However, enzyme-linked immunoassays can be problematic. because its activity depends on the temperature and instability of the enzyme or substrates may result in a inaccurate quantification of the target ligand. Other immunoassays control the fluorescence as the signal, with or without enzymes for the Measurement of analyte concentrations.

Transfer dyes of bi-fluorophores energy that provide a new methodology to control processes in biological systems. The fluorescent nature of such dyes allows them to control processes in which biological systems are involved. The signal fluorescent is measured by a fluorometer that connects to excite the fluorescent molecule at a specific wavelength and to measure fluorescence emission at another wavelength. The difference in excitation and emission of wavelengths is called Stokes shift.

Previously, various combinations of bifluorophores dyes have been described. US Patent No. 5,688,648, entitled "Probes Labelled with Energy Transfer Coupled Dyes" by Mathies et al ., Filed on December 19, 1995 describes sets of fluorescent markers carrying pairs of donor and acceptor dye molecules, where markers can be linked to nucleic acid skeletons for sequencing. A method for identifying and detecting nucleic acids in a multinucleic acid mixture using different fluorescent markers is included, where the fluorescent moieties are selected from families such as cyanine dyes or xanthenes. Fluorescent markers comprise pairs of fluorophores where a donor fluorophore has a mission spectrum that overlaps with the absorption of the acceptor fluorophore so that energy is transferred from the excited member to the other member of the pair.

U. S. Patent No. 2301 833 B entitled "Fluorescent Labelling Complexes with Large Stokes' Shifts Formed by Coupling Together Cyanina and Other Fluorochromes Capable of Resonance Energy Transfer" by Wagoner et al ., Filed May 30, 1996, describes complexes that they comprise a first fluorochrome that has first absorption and emission spectra and a second fluorochrome that has a second absorption and emission spectra. The linker groups between the fluorochromes are alkyl chains. The fluorescent nature of the dyes allows them to be useful in the sequencing and detection of nucleic acids.

EP 0805190 A2 refers to linkers to attach a donor dye to an acceptor dye in a dye fluorescent energy transfer and a fluorescent dye Energy transfer that has better fluorescence.

WO 97/11084 describes marks fluorescents that employ a combination of a component energy absorber / donor and an acceptor / fluorescent component of energy attached to a spacer comprising phosphate junctions of sugar without purine and pyrimidine bases that form a cassette.

In dye construction bi-fluorophore, are aspects of particular importance the distance between acceptor and donor molecules and the structure of the crimping groups. There is still the need for additional improvements in the construction of dyes, for example to accommodate the selection of biological molecules of various sizes

Summary of the invention

It has now been discovered that a new class of Energy transfer dyes are useful in materials marking involved in sequencing reactions and in other Applications. The dyes are preferably in the form of "cassettes" that allows them to join various materials Biological A cassette includes a bound structure or complex covalently with at least two traces of fluorescent dye, a crimp group and preferably a reactive group to join the complex to biological material or other target material. The group reagent is chosen because it is suitable to form a covalent bond with a functional group in a particular target material. Dyes are selected so that the emission spectrum of a dye overlaps the absorption spectrum of a second dye, allowing, therefore, that the transfer of energy takes place between two dyes Dye cassettes containing aryl crimp groups and heteroaryl provide stable and rigid structures to which biological molecules of various sizes can be attached, and which have features that allow the transfer of energy between fluorophores

The present invention provides dyes of energy transfer, reagents and methods defined in the independent claims.

Therefore, in one aspect, the present invention provides an energy transfer dye of the formula (I):

D_ {1} -L_ {1} -A-L2 -D_ {2}

in which D_ {1} is a first dye residue suitable as acceptor or donor in a transfer provision of energy; D_ {2} is a second dye that is suitable as donor or acceptor in an energy transfer provision with the first dye, or any additional dye added. TO It comprises 5-20 linked atoms, where the atoms linked contain at least and aryl or heteroaryl group and where atoms are independently selected from the compound group by carbon, nitrogen, sulfur, oxygen, phosphorus; L_ {1} includes  a group of 2 to 30 atoms bonded to bind said D1; L2 comprises a group of 2 to 30 atoms bonded to bind to said D2, and a functional group of 4 to 30 bonded atoms adapted to join A with a target biological

The chain can be optionally replaced, if you want, with groups known to those skilled in the art who they do not prevent the transfer of energy, for example, the ring it can be an aromatic or heteroaromatic ring substituted with one, two or three substituents independently selected from the group consisting of alkyl, alkoxy, alkynyl, alkenyl moieties, halogen, trihalomethyl, carboxylate, amino, nitro and ester or substituted with linear or branched C 1,2,3 or 4 alkyl, phenyl or arylalkyl, optionally substituted with 1, 2, 3 or 4 substituents independently selected from OH groups, halo, methyl, hydrogen or ethyl.

Additionally, L2 independently contains an atom or group selectively adapted for attachment to an additional linker to which a third energy transfer dye is attached in a cascade energy transfer arrangement, where the third dye interacts with the second dye (D 2) that interacts with the first dye (D 1), and an atom or group for binding to a target material, for example, a biological material as indicated more
ahead.

When D_ {1} or D_ {2} are pairs of fluorescein / rhodamine, there are probably 6 to 25 atoms of crimp joined together in A, L_ {{1} and L_ {2}], and more preferably 9, 10, 11, 12, 13, 14 or 15 crimping atoms. Preferably, A is an aromatic C 6 hydrocarbon moiety bonded to L_ {1} or L_ {2}. L_ {1} is a hydrocarbon chain C_ {2} -C_ {4} containing additional groups known to those skilled in the art that allow bonding to a molecule of xanthine or cyanine, and L2 is a chain of C4 hydrocarbon containing additional groups known for those skilled in the art that allow binding to a molecule of xanthine or cyanine and / or biological target material.

The specification of a range of values for several atoms in a chain or group, either an express list of each integer in the range indicated above, or a description of the interval specifying the endpoints of the interval, includes the specific description of each number value integer in such interval, including endpoints. Further, includes the specific description of each sub-interval within the larger interval For example, interval 1-6 includes subintervals 1-4 and 3-6, together with the other subintervals included.

The reagent or functional group, L2, can be any suitable group to join the transfer dye of energy to a target material, preferably a biological material Diana and, as such, will be known to specialists in the technique. Preferably, the L2 functional group for the binding to a target material is selected from the group consisting of carboxyl, succinimidyl ester, sulfo-succinimidyl ester, isothiocyanate, maleimide and phosphoramidite and groups covalently reactive with carboxyl, succinimidyl ester, sulfo-succinimidyl ester, isothiocyanate, maleimide and phosphoramidite.

The dyes suitable for D1 or D2 they can be dyes containing reagent or functional groups able to bind with L_ {1} or L_ {2}. The joining groups of L_ {1} and L_ {2} can be any suitable adaptation for connect to D_ {1} or D_ {2}. Preferably, the binding group Functional of L1 and / or L2 is PO3, NH-CO or NH-CS.

The dye residues, for example D1 or D2, of the present energy transfer dyes are fluorophores that are selected, as indicated later in this document, in order to participate in a provision of energy transfer

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Preferably, the transfer dyes of energy of this invention have a total molecular weight of less than 10,000 or 5,000 Daltons, more preferably less than 3,000 or 2,000 Daltons, still more preferably less than 1,500 or 1,200 Daltons

In relation to transfer dyes energy of the present invention, by "provision in energy transfer "means that two dyes fluorescents are selected having absorption and spectra of suitable emission for energy transfer between dyes, and are located with sufficient proximity and physical link in a way that the photoexcitation of a first dye (the donor) from place to transfer of energy from the first dye to the second dye (the acceptor). Energy transfers can also be created Additional involving one or more additional dye residues.

In this way, a "transfer dye of energy "refers to a complex of fluorescent dye that has at least two remains of dye that can participate in the energy transfer between these two dye residues, a cascading arrangement of energy transfer would imply, by therefore, more than two dye residues and at least three dyes that can participate in the transfer of energy between the three traces of dye

By "acceptor" in a provision of energy transfer means a remainder of dye that absorbs the energy in a wavelength emitted by a remainder of dye donor, that is, the absorption spectrum of the acceptor overlaps with the emission spectrum of the donor.

By "donor" in a provision of energy transfer means a remainder of dye that absorbs the energy of light, and that emits light at frequencies at least partially in the absorption spectrum of a dye residue acceptor

In the generic descriptions of compounds of this invention, the number of atoms of a particular type in a substituent group is generally provided in the form of interval. For example, an alkyl group containing from 1 to 4 carbon atoms Such interval reference attempts to include specific references for groups that have each of the numbers of atoms at specific intervals including end points Other members of atoms and other types of atoms are indicate in the same way, for example C_ {1-4} includes each of C 1, C 2, C 3 and C 4 individually and any subgroup of the interval.

Unless otherwise indicated, the term "alkyl" refers to an aliphatic hydrocarbon group branched or unbranched, which preferably has 1 to 6 carbon atoms, and more preferably 1 to 4 atoms of carbon. Preferably, the hydrocarbon group is saturated. The alkyl group may be optionally substituted, and some Preferred substituents include alkoxy, alkylthio, halogen, amino, monosubstituted amino, disubstituted amino and groups carboxy

The term "lower alkyl" refers to an aliphatic hydrocarbon having 1 to 6 carbons and preferably 1 to 4 carbon atoms. The alkyl group lower may be optionally substituted; the substituents Preferred include alkoxy, alkylthio, halogen, amino, amino monosubstituted, disubstituted amino and carboxy.

The term "branched alkyl" refers to to a branched aliphatic hydrocarbon. The branched alkyl group it is preferably 3 to 10 carbons, and more preferably 3 to 6 carbons The branched alkyl group may be substituted. optionally and some preferred substituents include alkoxy, alkylthio, halogen (such as fluorine, bromine, chlorine and iodine), amino, monosubstituted amino, disubstituted amino and carboxy.

The term "haloalkyl" refers to a lower alkyl group that is substituted with a halogen. This form, the term "fluoroalkyl" refers to a group lower alkyl which is substituted with a fluorine. The term "perfluoroalkyl" refers to a lower alkyl group that it is replaced with a fluorine atom in each available position except when the lower alkyl group joins the chain principal.

The term "aryl" refers to a group aromatic that has at least one ring that has a system of conjugated electron \ pi and includes both aryl groups carboxycyclics (eg, phenyl) as aryl groups heterocyclic (for example, pyridine).

Specific examples of heterocyclic groups Known in chemistry include the following:

one

two

The aryl group is preferably from 6 to 14 carbons, more preferably 6 to 10 carbons. Aryl remains include monocyclic, bicyclic, and tricyclic rings, where Each ring preferably has five or six members. The rest aryl may be monosubstituted or optionally substituted from independently with lower alkyl or alkenyl, alkynyl, hydroxyl, alkoxy, alkylthio, halogen, haloalkyl, mercapto, amino, monosubstituted amino or disubstituted amino.

The term "carbocyclic" refers to a compound or group containing one or more ring structures covalently closed, and that the atoms that form the skeleton of the Ring be all carbon atoms. In this way, the term distinguishes carbocyclic rings from heterocyclic rings in which The ring skeleton contains at least one non-carbon atom. A "cycloalkane" or "cyclic alkane" or "cycloalkyl" is a carbocyclic group in which the ring is a hydrocarbon optionally substituted cyclic aliphatic, that is, a group Cyclic alkyl preferably with 3, 4, 5 or 6 ring carbons. Thus, a "cyclopropyl" group has 3 atoms of carbon in the ring

By "branched or linear alkyl" is means a branched or chain saturated aliphatic hydrocarbon linear. Typical alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl and Similar. Halo means fluorine, chlorine, bromine or iodine.

In the context of this invention, the expression "target material" refers to a compound or structure to which an energy transfer dye covalently binds or to which such dye joins.

By "biological material" is meant a compound produced or present in an organism, including, but not limitation, polypeptides, nucleic acid molecules, Carbohydrates, and lipids. Such compounds can be adapted or derived to include a group suitable for covalent binding of An energy transfer dye. The expression does not mean that the dyes of the present invention should be used with organisms intact, since dyes will often be used with extracts, such as nucleic acid extracts, or samples, including samples conserved such as tissue sections, or in reactions of nucleic acid sequencing. Preferably, the size of the Biological material is between 400 mp and 1,000,000 mp. Plus preferably, the size is between 400 mp and 100,000 mp. and more preferably the size is between 500 and 15,000 mp.

Preferably, the donor dye is a dye of Xanthine or cyanine and the acceptor is a rhodamine or cyanine dye. Preferably, L1 and L2 contain a reactive group or Functional suitable for binding the dye to the corresponding component of the reactive or functional group of A. For example, for the binding of D1 to the chain L1, dyes are preferred that contain an activated carboxyl or carboxyl group. The choice of dyes that contain reactive and functional group that are suitable for the formation of covalent bonds with the chain L_ {1} and / or L_ {2} will be well known to specialists in the technique. Additionally, the choice of reactive and functional group for L_ {1} and / or L_ {2} suitable for link formation covalent with chain A will be well known to specialists in the technique

The appropriate xanthine donor dyes include, but not limited to, 5-carboxyfluorescein, 6-carboxyfluorescein, 6-carboxy-4'5'-dichloro-2'7'-dimethoxyfluorescein, CyA (3 - (ε-carboxyphenyl) -3'-ethyl-5,5'-dimethyl oxa-carboxycycline), Cy2 (3 - (ε-carboxyphenyl) -3'-ethyl-oxa-carbocyanine) and Cy3 (3 - (ε-carboxyphenyl) -l'-ethyl-3,3,3 ', 3'-tetramethyl-5,5'-disulfonate-carbocyanine.

Suitable cyanine dyes include, but are not limited to, Cy3.5 (3 - (? -Carboxyphenyl) -1'-ethyl-3,3,3 ', 3'-tetramethyl-4,5,4'5'- (1,3-disulfonate) dibenzo-carbocyanine), Cy5 (1 - (? -Carboxyphenyl) -l'-ethyl-3,3,3 ', 3'-tetramethyl-5,5'-disulfonate-dicarbocyanine, Cy5 .5 (1 - (? -Carboxypentyl) -1'-ethyl-3,3,3'3'-tetramethyl-4,5,4 ', 5' - (1,3-disulfonate) -dibenzodicarbocyanine, Cy7 ( 1- (? -Carboxypentyl) -1'-ethyl-3,3,3 ', 3'-tetramethyl-5,5'-disulfonate-tricarbocyanine) Cyanine dyes suitable for use in transfer dyes are described Energy of the present invention in US Patent No. 5,268,486 (Wagoner et al ., incorporated herein by reference in its entirety including any drawing).

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Suitable rhodamine acceptor dyes include, but not limited to; 5-carboxypylamine (Rhodamine 110-5), 6-carboxypylamine (Rhodamine 110-6), 5-carboxypylamine-6G (R6G-5 or REG-5), 6-carboxypylamine-6G (R6G-6 or REG -6), N, N, N ', N'-tetramethyl-5-carboxypylamine, N, N, N'N '' - tetramethyl-6-carboxypylamine (TAMRA or TMR), 5-carboxy-X-rhodamine, 6-carboxy-X-rhodamine  (ROX), Cy3 (3 - (ε-carboxyphenyl) -1'-ethyl-3,3,3 ', 3'-tetramethyl-5,5'-disulfonate-carbocyanine), Cy3.5 (3 - (? -Carboxypentyl) -1'-ethyl-3,3,3 ', 3'-tetramethyl-4,5,4', 5 '- (1,3-disulfotyl-5,5'-disulfonate ) dibenzocarbocyanine), Cy5 (1 - (? -Carboxypentyl) -1'-ethyl-3,3,3 ', 3'-tetramethyl-5,5'-disulfonatodicarbocyanine, Cy5.5 (1 - (? -Carboxypentyl) -1'-ethyl-3,3,3 ', 3'-tetramethyl-4,5,4', 5 '- (1,3-disulfonate) -dibenzo-dicarbocyanine and Cy7 (1 - (? -Carboxypentyl) -1'-ethyl-3,3,3 ', 3'-tetramethyl-5,5'-disulfonate-tricarbocyanine.

The above and additional dyes are described, for example, in Southwick et al ., 1990, Cytometry 11: 418-430; Mujumdar et al ., 1993, Bioconjugate Chemistry 4: 105-111; Wagoner and Ernest, Fluorescent Reagents for Flow Cytometry, Part 1; Principles of Clinical Flow Cytometry (1993) and Molecular Probes Handbook of Fluorescence Probes and Research Chemicals, Molecular Inc. 6th edition (1996) Haugland, which are all incorporated herein by reference in their entirety including any figure.

Optionally the complexes can contain a third dye, for example, a cyanine dye, bound to L2 through a suitable crimp group and that is in a provision of cascade energy transfer with D1 and D2.

By "provision of energy transfer in waterfall "means an energy transfer dye that It contains at least three dye residues. In this type of arrangement, the first dye is in an energy transfer arrangement with the second dye, where the second dye is the recipient of the energy transferred from the first dye. In addition, in this type of provision, the second dye is in a provision of energy transfer in which you receive a transfer of energy from the first dye and also transfers energy the third dye Preferably, an additional crimper is used for join the third dye to L2 {which is still available for join both D2 and a biological material.

In a further aspect, the present invention is refers to a biological material that contains a dye of energy transfer of the formula (I)

Suitable biological materials include, but are not limited to antibodies, antigen, peptides, proteins, carbohydrates, lipids, peptide nucleic acid nucleotides (PNAs), polynucleic oxy or deoxyribo, blocked nucleic acids (LNAs) as described in Kroshkin et al. , Tetrahedron Letters 1998, 39: 4381-4384 and cells that can be derived, if necessary, so that they contain one or more groups suitable for the binding of an energy transfer dye, for example, amino, hydroxy, thiophosphoryl groups, sulfydryl or carboxy.

In another aspect, the present invention provides an energy transfer dye of the formula (II):

3

Preferably, D1 in this type of arrangement serves as a donor dye and includes dyes selected from the groups composed of xanthine or cyanine. Previously suitable dyes of both xanthine have been analyzed like cyanine Additionally, in this type of arrangement, D_ {2} serves as a receiving dye and includes the selected dyes among the rhodamine or cyanine groups described above. Too it is preferred that the aryl ring can be substituted with chains saturated or unsaturated sides such as chains containing an ethylenic group (C = C) or chains containing a group acetylenic (C = C).

n1, n2 and n3 are a chain of linked atoms where atoms are selected from the group consisting of: carbon, oxygen, phosphorus, nitrogen and sulfur. Preferably, the chain is composed of hydrocarbons and carbonyls. Preferably n1, n2 and n3 are NH.

In a further aspect, the present invention provides an energy transfer dye of the formula (III):

4

Preferably, D1, in this type of arrangement, serves as a donor dye and includes dyes selected from the groups composed of xanthine or cyanine. Previously suitable xanthine dyes or cyanine Additionally, in this type of arrangement, D_ {2} serves dye receptor and includes selected dyes among the rhodamine or cyanine groups described above. Are included other embodiments as described for arrangement II.

In a further aspect, the present invention provides an energy transfer dye of the formula (IV):

5

Preferably, D1, in this type of arrangement, serves as a donor dye and includes dyes selected from the groups composed of xanthine or cyanine. Previously suitable xanthine dyes or cyanine Additionally, in this type of arrangement, D_ {2} serves receiving dye and includes the dyes selected between the groups rhodamine or cyanine described above. Others are included embodiments as described for arrangement II.

In a further aspect, the present invention provides an energy transfer dye of the formula (V):

6

Preferably, D1, in this type of arrangement, serves as a donor dye and includes dyes selected from the groups composed of xanthine or cyanine. Previously suitable xanthine dyes or cyanine Additionally, in this type of arrangement, D_ {2} serves dye receptor and includes selected dyes among the rhodamine or cyanine groups described above. Others are included additional embodiments as described for the arrangement II.

In a further aspect, the present invention provides an energy transfer dye of the formula (SAW):

7

\hbox{disposición II.}

Preferably, D1, in this type of arrangement, serves as a donor dye and includes the dyes selected from the groups consisting of xanthine or cyanine. Previously suitable xanthine or cyanine dyes have been analyzed. Additionally, in this type of arrangement, D 2 serves as a receptor dye and includes the dyes selected from the rhodamine or cyanine groups described above. The group "n_ {1}" is preferably 1 to 10 attached hydrocarbons, and in a further embodiment "n_ {") is equal to one. Other embodiments are included as described for the

 \ hbox {provision II.} 

In a further aspect, the present invention provides a method for preparing a dye from energy transfer according to claim 1 using preferably at least five coupling reactions:

(i) couple A with a component of L2;

(ii) couple the reaction product (i) with L_ {1} to be substituted with a reactive group suitable for form the junction with D1;

(iii) couple the reaction product (ii) with D1;

(iv) react the reaction product (iii) so that L2 is replaced with a suitable reactive group to form the union with D2 and a biological molecule;

(v) couple the product of (iv) with D2.

It will be easily apparent to specialists in the art that alternative coupling steps can be used for the same purpose An alternative, for example, would be to exchange D 1 with D 2 in stages i and iv.

In relation to the present dyes of energy transfer and the union of the various remains of these dyes, the term "couple" refers to the formation of a link or covalent bonds that link two components, for example, that bind a dye residue with the portion L_ {1} -A-L_ {2} of the dye energy transfer The expression "react" is refers to the addition or removal of groups or components that are they produce in the formation of a reactive group that can bind easily to a covalent binding molecule.

How can there be several reactive groups present in any component that participates in one of the coupling reactions, it may be necessary to block or protect those who do not participate in that reaction then check them out when appropriate at a later stage in the sequence of reactions.

The dye will usually contain a substituent suitable for the coupling reaction with the group L_ {1} -A-L_ {or} will be modified to contain such a group. For example, iodoacetamide is a Suitable substituent for xanthine dyes, maleimide is a Suitable substituent for cyanine dyes.

Fluorescent energy transfer dyes can be used to form reagents by covalently binding dyes to support materials such as polymer particles, cells, glass beads, antibodies, proteins, peptides, enzymes, carbohydrates, lipids and nucleotides or nucleic acids (DNA, PNA, LNA (Locked Nucleic Acids, "Novel convenient Synthesis of LNA [2.2.1] Bicyclo-Nucleosides", Koshkin et al ., Tetrahedron Letters 39: 4381-4384 (1998)) and RNA) and analogs containing or containing have derived to include at least one first reactive group capable of forming a covalent bond with the functional group in the labeling complex (or functional group capable of forming a covalent bond with a reactive group in the complex, as described above), and at least a second reactive group (or functional group, as the case may be), which has specificity for, and is capable of forming a covalent bond with, a target biological compound, such as antibodies, cells, drugs, antigen, bacteria, viruses and other microorganisms.

When the vehicle has functional groups, the functional groups may be antibody or DNA suitable for the binding to the antigen or a complementary DNA sequence, respectively. When the transport material has groups reagents, the vehicle can be a polymeric particle or a antigen suitable for binding to DNA or for example to a antibody. The techniques for fluorochromes that bind covalently to transport materials such as those mentioned they are well known in the art and are readily available in The bibliography

The transport material may also include derived nucleotides to contain one of the amino groups, sulfydryl, carboxyl, carbonyl or hydroxyl, and oxy acids or deoxy-ribo polynucleic or derivatized nucleic to contain one of the following amino groups, thiophosphoryl, sulfydryl, carboxyl, carbonyl or hydroxyl.

Functional groups in the support material which are complementary to, that is, capable of forming links covalent with the reactive groups of the labeling complexes of the invention include amino, carboxyl, carbonyl e groups hydroxyl

The present invention also relates to marking processes in which, in a first stage, a dye of energy transfer of the present invention reacts covalently with and mark, therefore, a first component and then use the first component marked to join with a second component to form a second marked component. So suitable, the first component may be a member of a pair of specific binding (a specific binding molecule). Then in the second stage of the procedure, the specific binding molecule fluorescently labeled is used as a test to join a second member of the specific binding pair (the second component) for the one with specific affinity.

Specific binding pairs may include a wide variety of molecules that are arbitrarily named ligands and receptors. An example of such pairs ligand-receptor includes an antibody and the corresponding antigen for which the antibody is specific. When the antibody target labeled in this way is a cell, the second stage of the procedure can be used to determine the amount of labeled antibodies that bind to such a cell type determining the intensity of the fluorescence of the cells. Through this procedure, monoclonal and other antibodies covalently marked components in a first stage with the fluorescent compounds of the present invention may be used as antigen testing

Those skilled in the art know others Numerous examples In this way, the additional pairs ligand-receptor include, for example, biotin- (strept) avidin, receptor hormonal-hormone, DNA-DNA complementary, DNA-RNA, DNA-binding protein, enzyme-substrate, and the like. It will be understood that Any two molecules with an affinity of specific bonding so that the energy transfer dyes of the present invention can be used to mark a member of a specific binding pair that in turn can be used in detection of the complementary member.

In a further embodiment, the present invention presents a method for determining the base sequence nucleotide of a DNA molecule composed of the stages of incubating a hybridized DNA molecule with a primer molecule able to hybridize with the DNA molecule in a container that Contains a thermostable DNA polymerase, a transfer dye of energy attached to a DNA sequencing terminator, where the binding is with a crimp of at least 5 and more preferably of at minus 10 atoms between the dye and the nucleotide, and separate the DNA products of the reaction and incubation according to the size by which at least a part of the base sequence Nucleotide of the DNA molecule can be determined. Such method is described in example III and Figures 7 and 8 provide data of sequences and a series of illustrations of the endings of dye energy transfer.

In preferred embodiments, the terminator of the Energy transfer dye is a compound selected from the group consisting of 5-FAM-Phe-5-REG-11-ddUTP: 5-FAM-Phe-5-REG-11-ddCTP: 5-FAM-Phe-5-REG-11-ddATP: 5-FAM-Phe-5-REG-11-ddGTP, 5-FAM-Phe-5-ROX-11-ddCTP, 5-FAM-Phe-5-ROX-11-ddUTP, 5-FAM-Phe-5-ROX-11-ddATP, 5-FAM-Phe-5-ROX-11-ddGTP, 5-FAM-Phe-5-TAMRA-11-ddGTP, 5-FAM-Phe-5-TAMRA-11-ddATP, 5-FAM-Phe-5-TAMRA-11-ddCTP, 5-FAM-Phe-5-TAMRA-11-ddUTP, 5-FAM-Phe-5-R110-11-ddATP, 5-FAM-Phe-5-R110-11-ddUTP, 5-FAM-Phe-5-R110-11-ddGTP  or 5-FAM-Phe-5-R110-11-ddCTP.

United States Patent Application titled "Didesoxi Dye Terminators", with the serial number 09 / 018,695 filed on February 4, 1998 and incorporated as a reference in this document in its entirety including any drawing, describes the importance of crimp length between dye and ddNTP. In the art the construction and union of various linkages. Reagents Suitable for linker construction include one or more compounds composed of activated forms of amino alkyl protected or aryl amino acids such as compounds of the formula R-NH- (CH 2) n -CO 2 R ' or R-NH- (CH2) n -X (CH2) m -CO_ {R} R ', in which R is a protecting group unstable acid or base, R 'is a reactive ester or group anhydride, X is aryl, O, S or NH and where n and m are 0-12. Other linkers are also suitable built by N- or O- or S-alkylation. The length exact crimp for a specific dye and combination dideoxynucleotide can be determined empirically by controlling the uniformity of the band in DNA sequencing.

Other features and advantages of the invention they will be evident from the following description of the preferred embodiments thereof and from the claims.

Brief description of the figures

Figure 1 is a reaction scheme for prepare the transfer dye terminator cassette energy (ET), N-5ROX-p-propargilamido-5FAM-L-phenylalanine-11-ddCTP (SFAM-ROX-Phe-11-ddCTP) and other terminators of the invention.

Figure 2 is a table of terminators of the blue ET dye.

Figures 3a, b and c are the data of the Chemical structure (3a) and Resonance Energy Transfer Fluorescence (3b, c) of ET Dye Terminator 5-FAM-Phe-5-ROX-11-ddCTP.

Figures 4a, b and c are the data of the Chemical structure (4a) and Resonance Energy Transfer Fluorescence (4b, c) of the ET Dye Terminator 5-FAM-Phe-5-REG-11-ddUTP.

Figures 5a, b and c are the data of the Chemical structure (5th) and Energy Transfer by Resonance Fluorescence (5b, c) of the ET Dye Terminator 5-FAM-Phe-5-TAMRA-11-ddGTP.

Figures 6a, b and c are the data of the Chemical structure (6a) and Resonance Energy Transfer Fluorescence (6b, c) of ET Dye Terminator 5-FAM-Phe-5-R1 10-ddATP.

Figure 7 is the sequencing data resulting from a DNA molecule using the dyes of Energy transfer described in this document.

Figure 8 is a set of four terminators of blue energy transfer dye.

The drawings are not necessarily to scale and certain features of the invention can be exaggerated in scale and be shown schematically in order to clarify and summarize.

Description of preferred embodiments

This invention relates to new dyes of energy transfer presenting energy transfer of fluorescence (transfer of energy-donor-acceptor). These New energy transfer dyes can be connected with excitation and emission wavelengths specific to facilitate a wide variety of tests or systems display. Assays such as molecular hybridization between residues such as nucleic acids or proteins, or for procedures such such as sequencing or component electrophoresis cell phones.

The energy transfer dyes of the present invention provide a valuable set of fluorescent markers that are particularly useful for multiparameter analysis and importantly are sufficiently low in molecular weight to allow materials labeled with the fluorescent complexes to penetrate cellular structures. As such, the dyes are suitable for use with DNA, PNA, LNA and / or RNA probes. Multiparameter analysis can be performed on multiple samples to detect the presence of target biological compounds. Each sample is marked by well known marking methods with an energy transfer dye. For example, a sample (sample 1) suspected of containing a target biological compound is incubated with a single fluorochrome such as fluorescein, Cascade Blue, a BODIPY dye, or one of the stiffened monometin dyes, or CY3 (SO3) 2, emitting all of them in wavelength ranges of 500-575 nm (green to orange). A second sample suspected of containing the target biological compound (the same compound or a different compound as in sample 1), is incubated with an energy transfer dye of the invention, for example N-5-tetramethyl-Rhodamine-p- Propargilamido-5FAM-L-phenylalaine, which will absorb light at 490 nm and emits fluorescence in the range of 525 nm-602 nm, depending on the Rhodamine dye selected. Additional samples suspected of containing another target compound are incubated with other dyes of the invention that have different absorbance and emission spectra than the dyes already used in the assay. After a suitable period to allow the fluorescent markers to bind with the target compounds, the unbound label is removed by washing and the labeled samples are
mix.

Detection is possible with a single source of wavelength excitation, i.e. 488 laser line nm. Each sample marked differently will emit fluorescence from a different color to the emission wavelength of its marker particular, allowing individual markers to be distinguished each.

Those skilled in the art will recognize that fluorescent energy transfer dial dyes of The present invention can be used for various techniques of immunofluorescence including direct and indirect immunoassays, and other known fluorescent detection methods. The conditions of each labeling reaction, for example, pH, temperature and time are known in the art, but generally room temperature is preferred. The pH is adjusted depending on optimal reaction conditions for reagent groups particular according to known techniques.

The energy transfer dyes of the present invention and reagents that can be made from they offer a wide variety of fluorescent markers with Great Stokes movements. Stokes displacements of dye should be as large as possible to minimize the noise measurement from the excitation source so that Maximize the relationship between signal and noise at the limit of sensitivity. The availability of dyes with displacements of Stokes above 100 nm is very limited. To limit additionally the usefulness of available dyes, the solubility of dyes in aqueous samples can be a problem because the most dyes with large stool movements are insoluble in water. The problem of a dye that has a Stokes small displacement is usually exceeded in the fluoromeditor technology through the use of agents monochromatic or expensive optical agents that filter the light from the source of excitement However, to overcome the loss of light intensity due to filters, for example, the use of high power light sources. These light sources produce heat that must dissipate in an instrument using heatsinks or thermal fans.

The incorporated fluorescent dye molecules in particles will present fluorescence inactivation due to the great proximity of the dyes to each other and to the matrix of the particle. The dyes are placed in the ET cassette at a distance of energy exchange that allows the transfer of energy donor-acceptor. In addition, the aryl crimp provides a rigid skeleton that adapts to provide a particular distance between fluorophores. Additionally, this crimping allows the attachment of the ET cassette to a variety more wide of biological molecules, varieties such as those described previously.

Those skilled in the art will appreciate that the dyes of the invention can be used in various applications of fluorescence over a wide range of the visible spectrum. Further, those skilled in the art will recognize that more than a couple of dye that presents fluorescence energy transfer can incorporated into molecules that give rise to a class of compounds They emit fluorescence at different wavelengths. Further, with the teachings of the invention described in this document, the incorporation into molecules of 3 or more dyes, which provide jointly a cascade of energy transfer from the absorbent to the intermediate donor and from this to the recipient (which issues fluorescence), can lead to the production of compounds with Stokes movements very long and allows to produce compounds with almost an unlimited variety of excitement and characteristics of issue.

Examples

The following examples serve to illustrate the Preparation of the energy transfer dyes herein invention. These examples do not attempt in any way to limit the scope of the invention.

Example 1 Preparation of the energy transfer cassette Rhodamine linked to phenylalanine FAM 1.1) Preparation of N-Boc- p -N-propargitrifluoroacetamido-L-phenylalanine

To a stirred suspension of N-Boc- p- iodo-L-phenylalanine (1.0 g, 2.55 mmol) and Cul (97 mg, 0.51 mmol., 0.2 equiv.) In anhydrous DMF (20 ml), in an Ar atmosphere, N-propargyltrifluoroacetamide (1.16 g, 7.67 mmol, 3 equiv), triethylamine (0.71 ml, 5.1 mmol, 2 equiv.) and tetrakis (triphenylphosphine) Pd were added (0) (295 mmol, 0.1 equiv.). The reaction was continued for 6 hours. Then, the mixture was diluted with 1: 1 MeOH-CH2Cl2 and treated with AGI X 8 resin (Bio-Rad) for 15 minutes, then filtered and the resin washed with 1: 1 of MeOH: CH 2 Cl 2. The combined filtrates were concentrated under reduced pressure and the residue obtained was absorbed on a silica gel for column chromatography. Elution with 5-15% MeOH in CHCl 3 gave N-Boc- p -N-propargyltrifluoroacetamido-L-phenylalanine, 1 H NMR (CD 3 OD) δ 7.31 (d, J = 6.7 Hz, 2H, Ar), 7.19 (d, J = 6.7 Hz, 2H, Ar), 6.49 (wide s, 1H, NH-Boc), 4.28 (wide s, 2H, CH2 -propargyl2), 4.21 (m, 1H, chiral CH), 2.84-3.25 (m, 2H, CH2 -phe), 1.38 (s , 9H, t-butyl).

1.2) Preparation of N-Boc- p -propargylamino-L-phenylalanine

To a stirred solution of N-Boc- p -N-propargyltrifluoroacetamido-L-phenylalanine (500 mg, 1.2 mmol) in MeOH (5 ml) was added 30% NH 4 OH and allowed to incubate for 4 hours. Then, the solution was evaporated under reduced pressure to give N-Boc- p -propargylamino-L-phenylalanine. 1 H NMR (DMSO-d 6) δ 7.35 (d, J = 6.7 Hz, 2H, Ar), 7.21 (d, J = 6.7 Hz, 2H, Ar ), 6.49 (broad s, 1H, NH-Boc), 4.00 (wide s, 2H, NH2), 3.75 (sa, 2H; CH2-propargyl), 3.48 (m, 1H, chiral CH), 2.81-3.55 (m, 2H, CH2 Phe), 1.32 (s, 9H, t-butyl).

1.3) Preparation of p -propargilamido-5FAM-L-phenylalanine

To stirred solution of N-Boc- p -propargylamino-L-phenylalanine (33 mg, 0.12 mmol) in anhydrous DMSO (3 ml) at room temperature under an Ar atmosphere was added di-isopropylethylamine (276 ml, 1 , 58 mmol, 15 equiv.) And 1 ml of a solution of DMSO and 5FAM-NHS ester (60 mg, 0.13 mmol, 1.2 equiv.). After 4 hours at room temperature, the reaction mixture was subjected to reverse phase column HPLC and eluted with a gradient of 0.05% aqueous TFA to 0.05% TFA in CH 3 CN for 30 minutes The fractions containing L-phenylalanine labeled with 5FAM were dried to provide N-Boc- p -propargyl amido-5FAM-L-phenylalanine. This compound was added to an ice-cold solution of 1: 1 aqueous TFA and stirred for 1 hour at room temperature to produce p-proparglylamide-SFAM-L-phenylalanine. 1 H NMR (CD 3 OD) δ 8.46 (s, 1H, Ar), 8.24 (d, J = 6.7 Hz, 1H, Ar), 6.52-7, 46 (m, 11H, Ar), 4.44 (s, 2H, CH2-propargyl), 4.25 (m, 1H, chiral CH), 3.06-3.19 (m, 2H, CH_ {2} -phe).

1.4) Procedure to convert p -propargilamido-5FAM-L-phenylalanine to N-5Rodamina- p -propargilamido-5FAM-L-phenylalanine (Figure 1)

p -Proparglylamide-5FAM-L-phenylalanine (15 mg, 0.03 mmol) was dissolved in anhydrous DMSO (2 ml) under an Ar atmosphere and stirred at room temperature. N, N'-di-isopropylethylamine (68 ml, 0.39 mmol, 15 equiv.) And 5-NHS solution of rhodamine-ester dye (0.39 mmpl, 1.2 equiv.) In DMSO was added anhydrous. After 1-2 hours, the solution was subjected to HPLC with a Delta Pak C18 reverse phase column (1.9 X 30 cm) and eluted with a gradient of 0.1 M TEAA to CH 3 CN at 20 ml / min for 30 minutes. The fractions containing the ET cassette were concentrated under reduced pressure.

Example 2 Preparation of the energy transfer dye attached to a dideoxynucleotide (5FAM-ROX-Phe-11-ddCTP) (Figures 1 and 3)

To a stirred solution of N-5ROX- p- proparglylamide-5FAM-L-phenylalanine was added N, N'-disuccinimidicarbonate and DMAP anhydrous THF solution. The corresponding NHS ester was formed after 20 minutes and the ester was conjugated with 5- (N-propargyl-6-aminocaproyl) ddCTP dissolved in buffer (pH 8.5). After stirring at room temperature for 1 hour, the solvent and buffer were removed under reduced pressure and the residue obtained was purified by vacuum column chromatography with SiO2 gel water aspirator eluting with 2: 8 to 1: 1 of MeOH in CHCl3 to MeOH, thus removing the free dye and impurities of N-hydroxysuccinimide. The fractions were further purified by reverse phase HPLC methods. The fluorescence enhancement of the energy transfer dye conjugate was shown to be 8.25 times that of the single ROX-labeled ddCTP.

It will be clear that using the examples above, others as biological molecules, such as deoxynucleotides and oligonucleotides, can bind to the corresponding NHS esters of the invention following the above methodology to produce compounds as illustrated in the drawings provided in this document.

Example 3 DNA sequencing using dideoxynucleoside triphosphates marked with the energy transfer dye (Figure 7)

A sequence of the M13mp18 DNA template was generated using a conventional "-40" primer. The reaction mixture (20 µl) contained 200 µM each of dATP, dCTP and dTTP, 1000 µM of dITP, 5-FAM-Phe-5-TAMRA-11-ddGTP 310 nM, 5-FAM-Phe -5-R110-11-ddATP 140 nM, 5-FAM-Phe-5-R6G-11-ddTTP 275 nM, 5-FAM-Phe-5-ROX-11-ddCTP 410 nM, 2 pmol primer 40, 200 ng of M13mp18 DNA, 20 units of Thermo Sequenase II (Amersham Pharmacia Biotech), 0.0008 units of inorganic pyrophosphatase from Thermoplasma acidophilum , 50 mM Tris-HCL pH 8.5, 35 mM KCl and 5 mM MgCl 2.

The reaction mixture was incubated in a cycler. 25 cycles of 95ºC, 30 sec; 60 ° C, 60 sec. After the cyclization, the reaction products were precipitated with ethanol using conventional procedures, washed, and they resuspended in buffer loaded with formamide. The sample was loaded on an Applied Biosystems model 377 instrument and the results are analyzed using conventional software methods.

It will be clear that using the examples above, others as energy transfer dyes of the invention, can be attached to the corresponding DNA terminators and used for sequencing reactions.

All patents and publications mentioned in Descriptive report indicates skill levels by those skilled in the art referred to in the invention. All references cited in this description are incorporate as a reference in the same scope as if each of this reference would have been incorporated as a reference in your totality individually.

A specialist in the art will appreciate easily that the present invention is well adapted for achieve the objectives and obtain the purposes and advantages mentioned, as well as the aspects inherent in this document. Dyes, substituents and target materials described herein presently representatives of preferred embodiments are illustrative and do not attempt to limit the scope of the invention. To the Technicians will come up with changes in these and other uses that are included within the spirit of the invention and are defined by the scope of the claims.

It will be readily apparent to a specialist in the technique made that several substitutions can be made and modifications of the invention described herein without get away from the scope and spirit of the invention. For example, the Technicians will easily recognize that those present Energy transfer dyes can incorporate several remnants of different dyes, linkers, binding groups and reactive groups and They can join several different target materials. This way such additional embodiments are within the scope of the present invention and of the claims that are included more ahead.

The invention described illustratively in This document can be properly practiced in the absence of any element or elements, limitation or limitations, that do not specifically described in this document. In this way, for example, in each case, in this document, any of the terms "understand", "consist essentially of" and "consist in "they can be replaced with some of the other two terms. The terms and expressions that have been used are used as terms of description and not of limitation, and do not attempt to exclude in use of such terms and expressions no equivalent of features shown and described or parts thereof, but it recognizes that several modifications are possible within the scope of the revindicated invention. In this way, it should be understood that although the present invention has been specifically described through preferred embodiments and optional features, modification and variation of concepts can be restored described in this document by specialists in the technique and that such modifications and variations are considered within the scope of this invention as defined by the attached claims.

In addition, when the characteristics or aspects of the invention are described in terms of Markush or other groups groups of alternatives, those skilled in the art will recognize that the invention is also described therefore in terms of any individual member or subgroup of group members Markush or another group.

In this way, the additional embodiments are within the scope of the invention and of the following claims.

Claims (26)

1. An energy transfer dye from the formula: D_ {1} -L_ {1} -A-L2 -D_ {2} in the what D_ {1} is a first remaining dye suitable as acceptor or donor in a transfer provision of Energy; D_ {2} is a second dye that is suitable as donor or acceptor in an energy transfer provision with the first dye; A comprises 5-20 atoms bonded, where bonded atoms contain at least one group aryl or heteroaryl and where atoms are selected independently between the group consisting of carbon, nitrogen, sulfide, oxygen, phosphorus; L1 comprises a group of 2 to 30 atoms linked to join said D1; L2 comprises a group of 2 to 30 atoms linked to join said D2, and a functional group of 4 to 30 bonded atoms adapted to join A with a target biological 2. The energy transfer dye of the claim 1, wherein L1 contains one or more rings aromatic 3. The energy transfer dye of the claim 2, wherein said aromatic ring is a ring phenyl. 4. The energy transfer dye of the claim 2, wherein L1 contains one or more rings heterocyclic 5. The energy transfer dye of the claim 1, wherein L2 contains one or more rings aromatic 6. The energy transfer dye of the claim 1, wherein A comprises a first chain of linear hydrocarbon bound to one or more heteroaryl groups, and said heteroaryl group also binds to a second chain of hydrocarbon. 7. The energy transfer dye of the claim 1, wherein D_ {1} is selected from the group composed of a xanthine dye and a cyanine dye, and D2 is selected from the group consisting of a rhodamine dye and a cyanine dye 8. The energy transfer dye of the claim 1, further comprising a third dye attached to L2 by means of a crimping group, where said union gives rise to an energy transfer arrangement of said third dye with D_ {2}. 9. The energy transfer dye of the claim 8 wherein said third dye is a dye of cyanine 10. A compound selected from the group composed of the formulas: 8 9

         \ vskip1.000000 \ baselineskip
      

10

         \ vskip1.000000 \ baselineskip
      

eleven

         \ vskip1.000000 \ baselineskip
      

12 where D_ {1} and D_ {2} are as defined in the claim one; n1, n2 and n3 are a chain of linked atoms where atoms are selected from the groups consisting of: carbon, oxygen, phosphorus, nitrogen, and sulfur; and n_ {1} is 1 a 10. 11. An energy transfer dye terminator selected from the group consisting of: 5-FAM-Pbe-5-REG-11-ddUTP: 5-FAM-Phe-5-REG-11-ddCTP: 5-FAM- Phe-5-REG-11-ddATP: 5-FAM-Phe-5-REG-11-ddGTP, 5-FAM-Phe-5-ROX-11-ddCTP, 5-FAM-Phe-5-ROX-11- ddUTP, 5-FAM-Phe-5-ROX-11-ddATP, 5-FAM-Phe-5-ROX-11-ddGTP, 5-FAM-Phe-5-TAMRA-11-ddGTP, 5-FAM-Phe- 5-TAMRA-11-ddATP, 5-FAM-Phe-5-TAMRA-11-
ddCTP, 5-FAM-Phe-5-TAMRA-11-ddUTP, 5-FAM-Phe-5-RI 10-11-ddATP, 5-FAM-Phe-5-R110-11-ddUTP, 5-FAM-Phe -5-R110-11-ddGTP or 5-FAM-Phe-5-R110-11-ddCTP. 12. A method to produce a dye from formula energy transfer: D_ {1} -L_ {1} -A-L2 -D_ {2} in the what D_ {1} is a first remaining dye suitable as acceptor or donor in a transfer provision of Energy; D_ {2} is a second dye that is suitable as donor or acceptor in an energy transfer provision with said first dye; A comprises 5-20 atoms bonded, where bonded atoms contain at least one group aryl or heteroaryl and where atoms are selected independently between the group consisting of carbon, nitrogen, sulfide, oxygen, phosphorus; L1 comprises a group of 2 to 30 atoms linked to join said D1; L2 comprises a group from 2 to 30 atoms bonded to bind said D2, and a group functional of 4 to 30 bonded atoms adapted to join A With a biological target. said method comprising: (a) coupling said A with a component of said L2; (b) couple the reaction product (a) with L1, where L1 is substituted with a suitable reactive group to form the union with D1; (c) couple the reaction product (b) with D1; (d) react the product of (c) so that L2 is replaced with a suitable reactive group to form the union with D2 and where a second part of L2 is replaced by a suitable reactive group to form the bond to a biological target, and (e) couple the product of (d) with D2, forming, therefore, said union. 13. A method for fluorescent marking a biological material comprising: (a) add to a liquid containing said biological material an energy transfer dye from the formula: D_ {1} -L_ {2} -A-L2 -D_ {2} D_ {1} is a first remaining dye suitable as acceptor or donor in a transfer provision of Energy; D_ {2} is a second dye suitable as a donor or acceptor in an energy transfer arrangement with said first dye; A comprises 5-20 atoms bonded, where bonded atoms contain at least one group aryl or heteroaryl and where atoms are selected independently between the group consisting of carbon, nitrogen, sulfide, oxygen, phosphorus; L1 comprises a group of 2 to 30 atoms linked; L2 comprises a group of 2 to 30 atoms linked to join said D2, and a functional group of 4 to 30 bonded atoms adapted to join A with a target biological; Y (b) react said dye so that said dye reacts covalently with and mark said material biological. 14. The method of claim 13, wherein said biological material is selected from the group consisting of antibodies, antigens, peptides, proteins, carbohydrates, lipids, nucleotides, oxy, deoxy, or deoxyribonucleic or polynucleic and cells that are optionally derived so that they contain one or more amino, hydroxy, thiophosphoryl, sulfydryl or carboxy groups. 15. A method for fluorescent marking a first component and then use said first marked component to detect the presence of a second component in a sample, which includes: (a) add a liquid containing said first Component an energy transfer dye of the formula: D_ {1} is a first remaining dye suitable as acceptor or donor in a transfer provision of Energy; D_ {2} is a second dye that is suitable as donor or acceptor in an energy transfer provision with said first dye; A comprises 5-20 atoms bonded, where bonded atoms contain at least one group aryl or heteroaryl and where atoms are selected independently between the group consisting of carbon, nitrogen, sulfide, oxygen, phosphorus; L1 comprises a group of 2 to 30 atoms linked; L2 comprises a group of 2 to 30 atoms linked to join said D2, and a functional group of 4 to 30 bonded atoms adapted to join A with a target biological (b) react said dye with said first component so that said dye reacts covalently and therefore mark said first component; (c) provide said first marked component to said sample to allow the attachment of said first component marking to said second component if present; Y (d) detect said second component if was present by detecting fluorescent labeling by optical method 16. The method of claim 15, wherein said target material comprises a biological material. 17. A reagent comprising: (a) an energy transfer dye from the formula: D_ {1} -L_ {2} -A-L2 -D_ {2} in the what D_ {1} is a first remaining dye suitable as acceptor or donor in a transfer provision of Energy; D_ {2} is a second dye suitable as a donor or acceptor in an energy transfer arrangement with said first dye; A comprises 5-20 atoms bonded, where bonded atoms contain at least one group aryl or heteroaryl and where atoms are selected independently between the group consisting of carbon, nitrogen, sulfide, oxygen, phosphorus; L1 comprises a group of 2 to 30 atoms linked; L2 comprises a group of 2 to 30 atoms linked to join said D2, and a functional group of 4 to 30 bonded atoms adapted to join A with a target biological; (b) a support material that contains or is has been derived to include at least one first reactive group capable of form a covalent bond with a functional group or group functional capable of forming a covalent bond with a reactive group in the energy transfer dye and covalently binds to the same. 18. The reagent of claim 17, wherein D_ {1} is selected from the group consisting of a dye of fluorescein and a cyanine dye, and said D2 is selected between the group consisting of a rhodamine dye and a dye of cyanine 19. The reagent of claim 17, wherein L2 is selected from the group consisting of amino, carboxyl, succinimidyl ester, sulfo-succinimidyl ester, isothiocyanate, maleimide and phosphoramidite and groups covalently reactive with carboxyl, succinimidyl ester, sulfo-succinimidyl ester, isothiocyanate, maleimide and phosphoramidite. 20. A biological material with a dye of United energy resonance, comprising: (a) an energy transfer dye from the formula: D_ {1} -L_ {2} -A-L2 -D_ {2} in the what D_ {1} is a first remaining dye suitable as acceptor or donor in a transfer provision of Energy; D_ {2} is a second dye suitable as a donor or acceptor in an energy transfer arrangement with said first dye; A comprises 5-20 atoms bonded, where bonded atoms contain at least one group aryl or heteroaryl and where atoms are selected independently between the group consisting of carbon, nitrogen, sulfide, oxygen, phosphorus; L1 comprises a group of 2 to 30 atoms linked; L2 comprises a group of 2 to 30 atoms linked to join said D2, and a functional group of 4 to 30 bonded atoms adapted to join A with a target biological; Y (b) a biological material that includes or has been derivative to include at least one first reactive group capable of form a covalent bond with a functional group, or a group functional capable of forming a covalent bond with a reactive group in the dye of energy transfer. and that covalently joins the same; 21. An energy transfer cassette of the formula: F1 --- NH ---

 \ delm {B} {\ delm {\ para} {M3}} 

--- D --- F2 in the what F 1 is a suitable first dye as an acceptor or donor in an energy transfer device; F 2 is a second dye that is suitable as donor or acceptor in an energy transfer provision with the first dye; B is the linear or branched alkyl chain that it contains 1 to 30 link atoms where said chain comprises one or more -NH -, - O -, - H = CH -, - C = C- and phenylenyl groups; M3 comprises hydrogen or a reagent or group functionally adapted to bind the energy transfer dye to a target material; Y D comprises an atom or group to bind F 2 to a set of crimp B. 22. The energy transfer dye of the claim 21, wherein F 1 is selected from the group composed of a xanthine dye and a cyanine dye and F2 is selected from the group consisting of a rhodamine dye and a cyanine dye 23. The energy transfer dye of the claim 21 further comprising a third dye attached to F 2 by a crimping group, where said bonding gives rise to to an energy transfer arrangement of said third dye with F 2. 24. An energy transfer dye from the formula: 13 in the what P1 is a suitable first dye as an acceptor or donor in an energy transfer provision; P2 is a second dye that is suitable as donor or acceptor in an energy transfer provision with the first dye; R 4 is hydrogen or alkyl C 1-4; R 5 comprises hydroxyl, halo, methyl or ethyl; N1 is a linker group containing 1 to 10 atoms or groups independently selected from carbon, nitrogen, oxygen, -CH = CH- and C = C-; S3 is hydrogen or a reagent or group functionally adapted to bind the energy transfer dye to A target 25. A method to determine the base sequence of nucleotides of a DNA molecule that comprises the steps from: (i) incubating a hybridized DNA molecule with a primer molecule capable of hybridizing to said DNA molecule in a vessel containing thermostable DNA polymerase, a compound of formula selected from the group consisting of 5-FAM-Phe-5-REG-11-ddUTP ,: 5-FAM-Phe-5-REG-11-ddCTP: 5-FAM-Phe-5-REG-11-ddATP: 5-FAM-Phe-5-REG-11-ddGTP, 5-FAM-Phe-5-ROX-11-ddCTP, 5-FAM-Phe-5-ROX-11-ddUTP, 5-FAM-Phe-5-ROX-11-ddATP, 5-FAM-Phe-5-ROX-11-ddGTP, 5-FAM-Phe-5-TAMRA-11-ddGTP, 5-FAM-Phe-5-TAMRA-11-ddATP, 5-FAM-Phe-5-TAMRA-11-ddCTP, 5-FAM-Phe-5-TAMRA-11-ddUTP, 5-FAM-Phe-5-R110-11-ddATP, 5-FAM-Phe-5-R110-11-ddUTP, 5-FAM-Phe-5-R110-11-ddGTP  or 5-FAM-Phe-5-R110-11-ddCTP. (ii) separate the DNA products from the reaction of incubation according to the size by which you can at least determining a part of the nucleotide base sequence of said DNA molecule; 26. A method to determine the presence of a or more molecules in a sample comprising the steps of: select specific binding molecules for at least one of said molecules in said sample, independently join each of these binding molecules to an individual energy transfer dye of claim 1, wherein D_ {1} is the same for each individual compound and D2 is different, mixing said binding molecules, bound together independently to individual compounds of formula 1 with said shows, determine the presence of said molecules based on different emission spectra.